Principal component and cluster analysis of morphological variables reveals multiple discrete sub-phenotypes in weaver mouse mutants.

The present study evaluates the usefulness of the principal component analysis-based cluster analysis in the categorization of several sub-phenotypes in the weaver mutant by using several morphological parameters from the cerebellar cortex of control, heterozygous (+/wv) and homozygous (wv/wv) weaver mice. The quantified parameters were length of the cerebellar cortex, area of the external granular layer, area of the molecular layer, number of the external granular layer cells (EGL), and number of Purkinje cells (PCs). The analysis indicated that at postnatal day 8, the genotype +/wv presented three sub-phenotypes tagged as +/wv (0), +/wv (1) and +/wv (2), whereas two sub-phenotypes designated as wv (0)/wv (1) and wv (0)/wv (2) were identified in the genotype wv/wv. The number of PCs for the genotype +/wv and the number of EGL cells for the genotype wv/wv were the variables that discriminated the best among sub-phenotypes. Each one of the sub-phenotypes showed specific abnormalities in the cytoarchitecture of the cerebellar cortex as well as in the foliar pattern. In particular, the wv (0)/wv (1) and wv (0)/wv (2) sub-phenotypes had the most altered cytoarchitectonics, followed by the +/wv (2) sub-phenotype and then by the +/wv (1) one. The sub-phenotype +/wv (0) was the less affected one. Apart from reporting for the first time the coexistence of several sub-phenotypes in the weaver mutant, our approach provides a new statistical tool that can be used to assess cerebellar morphology.

Laminar distribution of neurotransmitter receptors in different reeler mouse brain regions.

Mapping of multiple receptors of neurotransmitters provides insight into the spatial distribution of neurotransmission-relevant molecules in the cerebral cortex. During development, lack of reelin leads to impaired migration, disturbed lamination of the hippocampus and inverted neocortical layering. In the adult, reelin may regulate synaptic plasticity by modulating neurotransmitter receptor function. Using quantitative in vitro receptor autoradiography, different receptors, in particular, the binding site densities and laminar distribution of various glutamate, GABA, muscarinic and nicotinic acetylcholine, serotonin, dopamine and adenosine receptors, were analyzed in cortical and subcortical structures of reeler and wild-type brains. Differential changes in the laminar distribution, maximum binding capacity (B (max)) and regional density of neurotransmitter receptors were found in the reeler brain. A decrease of whole brain B (max) was found for adenosine A(1) and GABA(A) receptors. In the forebrain, several binding sites were differentially up- or down-regulated (kainate, A(1), benzodiazepine, 5-HT(1), M(2), α(1) and α(2)). In the hippocampus, a significant decrease of GABA(B), 5-HT(1) and A'1 receptors were observed. The density of M(2) receptors increased, while other receptors remained unchanged. In the neocortex, some receptors demonstrated an obviously inverted laminar distribution (AMPA, kainate, NMDA, GABA(B), 5-HT(1), M(1), M(3), nAch), while the distribution of others (A(1), GABA(A), benzodiazepine, 5-HT(2), muscarinic M(2), adrenergic α(1), α(2)) seemed to be less affected. Thus, the laminar receptor distribution is modulated by the developmental impairment and suggests and reflects partially the laminar inversion in reeler mice.

Regional differences in the vulnerability of substantia nigra dopaminergic neurons in weaver mice.

Vulnerability of midbrain dopaminergic (DA) neurons in the weaver mouse was studied at postnatal (P) days 8 and 90, in chosen coronal levels throughout the anteroposterior (AP) extent of the substantia nigra pars compacta (SNc). Wild-type (+/+) and homozygous weaver (wv/wv) mice used were the offspring of pregnant dams injected in several cases with tritiated thymidine on embryonic days 11-15. DA neurons were identified for their tyrosine hydroxylase immunoreactivity. Data reveal that at P8, the frequency of both +/+ and wv/wv late-generated DA cells increases from rostral to caudal SNc. No apparent DA-cell loss was observed at P8 in the mutant genotype, irrespective of the AP level considered. However, throughout the AP, there was a significant reduction in the number of these neurons at any level in 90-day-old weavers. Comparison of P8 and P90 +/+ SNc suggests that cell death is not a major aspect in the developmental regulation of normal DA neurons, although numerical cell depletion in the postnatal development of weaver SNc probably results from the amplification of a basal cell-death process, which affected all the coronal levels studied.

Developmental anatomy of reeler mutant mouse.

The reeler mouse is one of the most famous spontaneously occurring mutants in the research field of neuroscience, and this mutant has been used as a model animal to understand mammalian brain development. The classical observations emphasized that laminar structures of the reeler brain are highly disrupted. Molecular cloning of Reelin, the gene responsible for reeler mutant provided insights into biochemistry of Reelin signal, and some models had been proposed to explain the function of Reelin signal in brain development. However, recent reports of reeler found that non-laminated structures in the central nervous system are also affected by the mutation, making function of Reelin signal more controversial. In this review, we summarized reported morphological and histological abnormalities throughout the central nervous system of the reeler comparing to those of the normal mouse. Based on this overview of the reeler abnormalities, we discuss possible function of Reelin signal in the neuronal migration and other morphological events in mouse development.

Formation and maturation of parallel fiber-Purkinje cell synapses in the Staggerer cerebellum ex vivo.

In vivo, homozygous staggerer (Rora(sg/sg)) Purkinje cells (PCs) remain in an early stage of development with rudimentary spineless dendrites, associated with a lack of parallel fiber (PF) input and the persistence of multiple climbing fibers (CFs). In this immunocytochemical study we used cerebellar organotypic cultures to monitor the development of Rora(sg/sg) PF-PC synapses in the absence of CF innervation. Ex vivo the vesicular glutamate transporters VGluT1 and VGluT2 reactivity was preferentially localized around the Rora(sg/sg) PC soma and proximal dendrites, which are typically CF domains. The shift from VGluT2 to VGluT1 in PF terminals during development was delayed in Rora(sg/sg) slices. The postsynaptic receptors mGluR1 and GluRdelta2 were differently distributed on Rora(sg/sg) PCs. mGluR1 reactivity was evenly distributed in PC soma and dendrites, whereas GluRdelta2 reactivity, normally restricted at PF synapses, was dense in Rora(sg/sg) PC somata. The presynaptic distribution of VGluT1 and VGluT2 on Rora(sg/sg) PCs matched the postsynaptic distribution of the glutamate receptor GluRdelta2, but not mGluR1.

Unusual patch-matrix organization in the retrosplenial cortex of the reeler mouse and Shaking rat Kawasaki.

The rat granular retrosplenial cortex (GRS) is a simplified cortex, with distinct stratification and, in the uppermost layers, distinct modularity. Thalamic and cortical inputs are segregated by layers and in layer 1 colocalize, respectively, with apical dendritic bundles originating from neurons in layers 2 or 5. To further investigate this organization, we turned to reelin-deficient reeler mouse and Shaking rat Kawasaki. We found that the disrupted lamination, evident in Nissl stains in these rodents, is in fact a patch-matrix mosaic of segregated afferents and dendrites. Patches consist of thalamocortical connections, visualized by vesicular glutamate transporter 2 (VGluT2) or AChE. The surrounding matrix consists of corticocortical terminations, visualized by VGluT1 or zinc. Dendrites concentrate in the matrix or patches, depending on whether they are OCAM positive (matrix) or negative (patches). In wild-type rodents and, presumably, mutants, OCAM(+) structures originate from layer 5 neurons. By double labeling for dendrites (filled by Lucifer yellow in fixed slice) and OCAM immunofluorescence, we ascertained 2 populations in reeler: dendritic branches either preferred (putative layer 5 neurons) or avoided (putative supragranular neurons) the OCAM(+) matrix. We conclude that input-target relationships are largely preserved in the mutant GRS and that dendrite-dendrite interactions involving OCAM influence the formation of the mosaic configuration.

Purkinje cell age-distribution in fissures and in foliar crowns: a comparative study in the weaver cerebellum.

Generation and settling of Purkinje cells (PCs) are investigated in the weaver mouse cerebellum in order to determine possible relationships with the fissuration pattern. Tritiated thymidine was supplied to pregnant females at the time that these neurons were being produced. Autoradiography was then applied on brain sections obtained from control and weaver offspring at postnatal (P) day 90. This makes it possible to assess the differential survival of neurons born at distinct embryonic times on the basis of the proportion of labeled cells located at the two foliar compartments: fissures and foliar crowns. Our data show that throughout the surface contour of the vermal lobes, generative programs of PCs were close between wild type and homozygous weaver. Similar data were found in the lobules of the lateral hemisphere. On the other hand, the loss of PCs in weaver cerebella can be related to foliar concavities or convexities depending on the vermal lobe or the hemispheric lobule studied. Lastly, we have obtained evidence that late-generated PCs of both normal and mutant mice were preferentially located in fissures. These quantitative relationships lead us to propose a model in which the final distribution of PCs through the vermal contour would be coupled to two factors: the cortical fissuration patterning and a "time-sequential effect" of weaver mutation.

Regional variations of cytochrome oxidase activity in the central auditory system of Relnrl-Orl (reeler) mutant mice.

Despite preserved cell differentiation, the Reln(rl-Orl) phenotype comprises laminar abnormalities of cell position in auditory cortex and dorsal cochlear nucleus. The metabolic consequences of the cell ectopias were determined by estimating cytochrome oxidase (CO) activity, a marker of neuronal activity. CO activity increased in the granular cell layer of dorsal cochlear nucleus, trapezoid body nucleus, intermediate lateral lemniscus, central and external inferior colliculus, and pyramidal cell layer of primary auditory cortex. On the contrary, CO activity decreased in the superficial molecular layer of dorsal cochlear nucleus as well as in the medioventral periolivary nucleus. These metabolic variations are discussed in terms of their possible relation to morphologic anomalies observed in the mutant.

The anatomy of the cerebellar nuclei in the normal and scrambler mouse as revealed by the expression of the microtubule-associated protein kinesin light chain 3.

Conventional kinesin is a motor protein complex including two heavy chains and two light chains (KLC). Junco et al. (Junco, A., Bhullar, B., Tarnasky, H.A. and van der Hoorn, F.A., 2001. Kinesin light-chain KLC3 expression in testis is restricted to spermatids. Biol. Reprod. 64, 1320-1330). recently reported the isolation of a novel KLC gene, klc3. In the present report, immunohistochemistry has been used to characterize the expression of KLC3 in the cerebella of normal and scrambler (scm) mutant mice. In cryostat sections through the cerebellum of the normal adult mouse immunoperoxidase stained for KLC3, reaction product is deposited in the nuclei and somata of deep cerebellar nuclear neurons. No other structures are stained in the cerebellum. Strong and specific KLC3 expression is observed in the adult cerebellum in all three major cerebellar nuclei--medial, interposed, and lateral. Double immunofluorescence studies reveal that KLC3 immunoreactivity is colocalized with both endosomes and GW bodies. KLC3 immunohistochemistry has been exploited to study the organization of the cerebellar nuclei in scrambler mice, in which disruption of the mdab1 gene results in severe foliation defects due to Purkinje cell ectopia, with most Purkinje cells clumped in centrally located clusters. Despite the severe failure of Purkinje cell migration, the cerebellar nuclei appear normal in scrambler mutant mice, suggesting that their topography is dependent neither on normal Purkinje cell positioning nor the Reelin signaling pathway.

The Purkinje cell degeneration (pcd) mouse: an unexpected molecular link between neuronal degeneration and regeneration.

The spontaneous autosomal recessive mouse mutation, Purkinje cell degeneration (pcd), was first identified through its ataxic behavior. Since its discovery in the 1970s, the strain has undergone extensive investigation, although another quarter century elapsed until the mutant gene (agtpbp1 a.k.a. Nna1) underlying the pcd phenotype was identified. As Nna1 was initially discovered as a gene induced in motor neurons following axotomy the finding that its loss leads to selective neuronal degeneration points to a novel and unexpected common molecular mechanism contributing to the apparently opposing processes of degeneration and regeneration. The elucidation of this mechanism may of course have significant implications for an array of neurological disorders. Here we will first review the principle features of the pcd phenotype and then discuss the functional implications of more recent findings emanating from the characterization of Nna1, the protein that is lost in pcd. We also provide new data on the genetic dissection of the cell death pathways operative in pcd(3J) mice, proving that granule cell death and Purkinje cell death in these mice have distinct molecular bases. We also provide new information on the structure of mouse Nna1 as well as Nna1 protein levels in pcd(3J) mice.

Expression of amino acid receptors and neural peptides in the weaver mouse brain.

In the present study, we conducted: (i) in situ hybridization in order to investigate the expression of kainate and GABA(A) receptor subunits and the pre-proenkephalin and prodynorphin peptides in the brain of weaver mouse (a genetic model of dopamine deficiency) and (ii) immunocytochemistry in order to study the somatostatin-positive cells in weaver striatum. Our results indicated: (i) increases in mRNA levels of KA2 and GluR6 kainate receptor subunits, of alpha(4) and beta(3) GABA(A) receptor subunits and of pre-proenkephalin and prodynorphin in 6-month-old weaver striatum; (ii) a decrease in alpha(1) and beta(2) GABA(A) subunit mRNAs in 6-month-old weaver globus pallidus; (iii) increases in KA2, alpha(4) and beta(3) and decreases in alpha(2) and beta(2) mRNAs in the 6-month-old weaver somatosensory cortex; and (iv) an increase in somatostatin-immunopositive cells in 3-month-old weaver striatum. We suggest that: (i) in striatum, the alterations are induced by the induction of the transcription factor DeltafosB (for GluR6, pre-proenkephalin and prodynorphin mRNAs) and the suppression of transcription factors like NGF-IB (nerve growth factor inducible B; for the KA2 mRNA), in response to dopamine depletion; (ii) in striatum and cortex, the alterations in the expression of the GABA(A) subunits indicate an increase of extrasynaptic versus a decrease of synaptic GABA(A) receptors; and (iii) in globus pallidus, the increased striatopallidal GABAergic transmission leads to a decrease in the number of GABA(A) receptors. Our results further clarify the regulatory role of dopamine in the expression of amino acid receptors and striatal neuropeptides.

The Lurcher mouse: fresh insights from an old mutant.

The Lurcher mouse was first discovered in 1954 as a spontaneously occurring autosomal dominant mutation that caused the degeneration of virtually all cerebellar Purkinje cells and most olivary neurons and granule cells. More recent molecular studies revealed that Lurcher is a gain of function mutation in the delta2 glutamate receptor (GluRdelta2) that converts an alanine to threonine in the highly conserved third hydrophobic segment of GluRdelta2. The mutation converts the receptor into a constitutively leaky cation channel. The GluRdelta2 receptor is predominantly expressed in cerebellar Purkinje cells and in the heterozygous Lurcher mutant (+/Lc). Purkinje cells die due to the mutation in the GluRdelta2 receptor, while olivary neurons and granule cells degenerate due to the loss of their Purkinje cell targets. The purpose of the review is to provide highlights from 5 decades of research on the Lurcher mutant that have provided insights into the developmental mechanisms that regulate cell number during development, cerebellar pattern formation, cerebellar physiology, and the role of the cerebellum in CNS function.

Abnormal laminar position and dendrite development of interneurons in the reeler forebrain.

The majority of cortical and hippocampal interneurons originate in the subcortical telencephalon and migrate tangentially into pallial regions before settling in various cortical layers. The molecular cues that regulate final positioning of specific interneurons in cortical structures have not yet been identified. The positioning of radially migrating principal neurons of the cortex and hippocampus depends upon Reelin, an extracellular protein expressed near the pial surface during embryonic development that is absent in reeler mutant mice. To determine whether the layer specification of interneurons, like that of principal neurons, requires Reelin, we crossed reeler with transgenic mice that contain Green Fluorescent Protein (GFP)-expressing Inhibitory Neurons (GINs). These neurons express basal forebrain markers Dlx1/2 in normal and reeler mice. In normal mice, GINs express Reelin and are localized to specific layers of the cortex and hippocampus. In reeler mutant mice, we show that GINs migrate normally into the pallium, but fail to acquire proper layer position. Double labeling experiments indicate that the neurochemical profile of these interneurons is not generally altered in reeler mice. However, the extension of their cellular processes is abnormal. Quantitative analysis of GINs in the cortex revealed that they are hypertrophic, bearing longer neuritic branches than normal. Thus, the lack of Reelin signaling results in abnormal positioning and altered morphology of forebrain interneurons.

The superficial layers of the superior colliculus are cytoarchitectually and myeloarchitectually disorganized in the reelin-deficient mouse, reeler.

The causative gene for the reeler mouse is reelin which encodes Reelin protein, an extracellular molecule. In the present study, we have examined the cytoarchitecture, myeloarchitecture, and afferent/efferent systems of the superior colliculus (SC) of the reeler mouse. In the reeler, the laminar structures of the superficial three layers of the SC were disorganized and intermingled into a single layer, i.e., the superficial fused layer (SuF), as previously reported in the reelin-deficient SRK rat (Sakakibara et al., Develop. Brain Res. 141:1-13). Next, we have investigated the course and terminals of visual corticotectal and retinotectal projections with an injection of biocytin into the visual cortex or an injection of cholera toxin subunit B into the retina, respectively. In the reeler, anterogradely labeled visual corticotectal and retinotectal fibers took an aberrant course within the SuF, resulting in abnormal myeloarchitecture of the superficial SC of the reeler. Retrograde labeling of tectospinal tract neurons could not show any differences between the normal and reeler mice, suggesting that the deep layers of the reeler SC are cytoarchitectually normal. In situ hybridization and immunohistochemical studies have shown that reelin mRNA and Reelin protein were both recognized in the normal SC. These results suggest that Reelin protein plays some roles in histogenesis of the superficial layers of the SC.

Neuroanatomical phenotypes in the reeler mouse.

The reeler mouse (Reln) has been proposed as a neurodevelopmental model for certain neurological and psychiatric conditions and has been studied by qualitative histochemistry and electron microscopy. Using magnetic resonance microscopy (MRM), we have quantitated for the first time the neuromorphology of Reln mice at a resolution of 21.5 microm. The neuroanatomical phenotypes of heterozygous and homozygous mutant Reln mice were compared to those of wild type (WT) littermates using morphometry and texture analysis. The cortical, hippocampal, and cerebellar phenotypes of the heterozygous and homozygous mutant Reln mice were confirmed, and new features were revealed. The Reln(rl/rl) mice possessed a smaller brain, and both Reln(rl/+) and Reln(rl/rl) mice had increased ventricles compared to WT controls. Shape differences were found between WT and Reln(rl/rl) brains, specifically in cerebellum, olfactory bulbs, dorsomedial frontal and parietal cortex, certain regions of temporal and occipital lobes, as well as in the lateral ventricles and ventral hippocampus. These findings suggest that certain brain regions may be more severely impacted by the Reln mutation than others. Gadolinium-based active staining demonstrated that layers of the hippocampus were disorganized in Reln(rl/rl) mice and differences in thickness of these layers were identified between WT and Reln(rl/rl) mice. The intensity distributions characteristic to the dorsal, middle, and ventral hippocampus were altered in the Reln(rl/rl), especially in the ventral hippocampus. These differences were quantified using skewness and modeling the intensity distributions with a Gaussian mixture. Our results suggest that structural features of Reln(rl/rl) brain most closely phenocopy those of patients with Norman-Roberts lissencephaly.

Purkinje neuron degeneration in nervous (nr) mutant mice is mediated by a metabolic pathway involving excess tissue plasminogen activator.

Purkinje neurons (PNs), the central cells in cerebellar circuitry and function, constitute a vulnerable population in many human genetic, malignant, hypoxic, and toxic diseases. In the nervous (nr) mutant mouse, the majority of PNs die in the fourth to fifth postnatal weeks, but the responsible molecules are unknown. We first disclose a remarkable increase in mRNA expression and protein concentration in the nr cerebellum of tissue plasminogen activator (tPA), a gene closely linked to the mapped but as-yet-uncloned nr locus. Evidence that excessive tPA triggers nr PN death was obtained with organotypic slice cultures expressing the nr PN phenotype, in which an inhibitor of tPA led to increased nr PN survival. An antagonist of protein kinase C, a downstream component in the tPA pathway, also increased nr PN survival. Additional downstream targets in the tPA pathway (the mitochondrial voltage-dependent anion channel, brain-derived neurotrophic factor, and neurotrophin 3) were also abnormal, in parallel with the alterations in PN mitochondrial morphology, dendritic growth, and synaptogenesis that culminate in nr PN death and motor incoordination. We thus propose a molecular pathway by which the excessive tPA in nr cerebellum mediates PN degeneration.

Moving up or moving down? Malpositioned cerebellar unipolar brush cells in reeler mouse.

Cerebellar morphogenesis occurs through a complex interplay of cell proliferation and migration that in mouse and rat begins about midgestation and ends in the third postnatal week. Cerebellar cells derive from germinative matrices in the ventricular zone and the external granular layer. Like granule cells, unipolar brush cells (UBCs) are excitatory interneurons situated in the granular layer of the cortex and innervated by mossy fibers. While granule cells are produced from the external granular layer, the generation of UBCs is still controversial. We utilized the reeler mutant mouse, which has widespread misplacement of neurons due to lack of Reelin protein, to ascertain the origin of UBCs. In the reeler cerebellum, which is small and lacks foliation, Purkinje cells are greatly reduced in number and in large part are located ectopically in deep cerebellar masses. Granule cells are also reduced in number and form an irregular granule cell layer. In this study we demonstrate that the reeler mutation influences the positioning of UBCs and also significantly reduces their number. Both subsets of UBCs identified in normal mouse, the calretinin-positive and the metabotropic glutamate receptor 1alpha-positive subsets, are affected in the reeler. About 40% of the calretinin-positive UBCs are ectopically situated in the deep cerebellar regions and the immediate vicinity of the ependyma of the fourth ventricle. Ectopic UBCs have discrete, although somewhat looser brushes than granular layer UBCs, but form synaptic junctions with complex axon terminals, possibly belonging to mossy fibers and UBC axons, like their normally situated counterpart. The observed displacement of UBCs in the reeler suggests that they originate from the ventricular zone.

Gaba- and serotonin-immunoreactive structures and ca(2+)-binding protein in the neocortex of the reeler mouse mutant.

The transmitter organization of anomalously formed neocortex was studied in reeler mutant mice by immunohistochemical studies of GABA- and serotoninergic structures and Ca(2+)-binding protein. GABAergic structures were identified in terms of the localization of glutamate decarboxylase (GDC) within them, this being an enzyme involved in GABA synthesis. The neocortex of reeler mutant mice was found to contain an unusual distribution of cells morphologically and chemically identical to Cajal-Retzius cells - beneath layer I rather than in its upper third, as seen in normal animals. GDC-immunoreactive label accumulated in the neuropil of the intermediate and deep layers, layer I containing only occasional granules. Serotonin-immunoreactive fibers did not form superficial or deep plexuses, as seen in normal animals, though they did reach their innervation targets. Thus, the anomalously formed neocortex which lacks the typical cytoarchitectonic organization, showed abnormalities in the structure of both intrinsic and projectional transmitter systems.

Compartmentation of the reeler cerebellum: segregation and overlap of spinocerebellar and secondary vestibulocerebellar fibers and their target cells.

The cerebellum of the reeler mutant mouse has an abnormal organization; its single lobule is composed of a severely hypogranular cortex and a central cerebellar mass (CCM) consisting of Purkinje cell clusters intermixing with the cerebellar nuclei. As such the reeler represents an excellent model in which to examine the effect of the abnormal distribution of cerebellar cells on afferent-target relationships. To this effect we studied the organization of the spinocerebellar and secondary vestibulocerebellar afferent projections in homozygous reeler mice (rl/rl) using anterograde tracing techniques. Spinal cord injections resulted in labeled spinocerebellar mossy fiber rosettes in specific anterior and posterior regions of the cerebellar cortex. Some vestiges of parasagittal organization may be present in the anterior projection area. Within the CCM, labeled fibers appeared to terminate on distinct groups of Purkinje cells. Thus, the spinocerebellar mossy fibers seem to form both normal and heterologous synapses in the reeler cerebellum. Secondary vestibular injections resulted in both retrograde and anterograde labeling. Retrograde labeling was seen in clusters of Purkinje cells and cerebellar nuclear cells; anterograde labeling was distributed in the white matter and in specific regions of the anterior and posterior cortex of the cerebellum. The labeled spinocerebellar and secondary vestibulocerebellar afferents overlapped in the anterior region but in the posterior region the vestibulocerebellar termination area was ventral to the spinocerebellar area. An area devoid of labeled terminals was also observed ventral to the posterior secondary vestibulocerebellar termination field. Using calretinin immunostaining it was determined that this area contains unipolar brush cells, a cell type found primarily in the vestibulocerebellum of normal mice. Our data indicate that despite of the lack of known landmarks (fissures, lobules) the spinocerebellar and vestibulocerebellar afferent projections in the reeler cerebellum do not distribute randomly but have specific target regions, and the position of these regions, relative to each other, appears to be conserved. Two caveats to this were the finding of overlapping terminal fields of these afferents in the anterior region, and a posteroventral region that contains unipolar brush cells yet is devoid of secondary vestibulocerebellar afferents. The distribution of Purkinje cells and cerebellar nuclear cells is not random either; those that give rise to cerebellovestibular efferents form distinct groups within the central cerebellar mass.